The following viruses are examples of viruses that are propagated in embryonated eggs or in primary cultures of chicken embryo fibroblasts prepared from embryonated eggs: avian influenza virus, avian reovirus, fowlpox virus, psittacine herpesvirus (Pachecco's herpesvirus), swine influenza virus, equine influenza virus, Newcastle disease virus, falcon herpesvirus, pigeon herpesvirus, infectious bursal disease virus, infectious bronchitis virus, Marek's disease virus, turkey herpesvirus, chicken anemia virus, avian encephalomyelitis virus, pigeon pox virus, canary pox virus, quail pox virus, avian polyomavirus types I and II, and avian adenovirus types I, II, and III. Detection of any one of these viruses as the etiological agent for a diseased animals can be determined by serological based assays or unequivocally by virus isolation (VI) diagnostic assays. For many of these viruses, live or killed vaccines are available to protect the animal from infection by the aforementioned viruses.
Vaccines comprising any of the aforementioned viruses are prepared by infecting embryonated chicken eggs or primary cultures of chicken embryo fibroblast (CEF) cells or a mammalian cell line in the case of infectious bursal disease virus (P. Van Der Marel and P. G. Mooren, U.S. Pat. No. 5,192,539). However, primary CEF cell cultures or embryonated eggs suffer from the disadvantage that theses substrates are prone to contamination with not easily detected viruses of which the probability cannot always be established prior to the actual use. Therefore, purity of vaccines prepared using these substrates cannot be established until after the preparation of the vaccine. Primary cell cultures and embryonated eggs depend on a reliable supply of embryonated eggs which can be obtained from commercial suppliers that maintain specified pathogenfree (SPF) flocks of chickens, chickens completely free of viral and bacterial contamination. However, it is difficult to continually maintain SPF flocks completely free of infection which is evidenced by the periodic outbreaks of disease in SPF flocks. The cost of SPF embryonated eggs, the cost of preparing CEF cells from embryonated eggs, and the cost of maintaining SPF flocks of chickens is expensive and reflects approximately 40% of the cost of vaccines prepared from embryonated eggs or CEF cells. Additional costs are incurred by vaccine manufacturers producing vaccines comprising any of the aforementioned viruses. Vaccine manufacturers must hold from sale every vaccine lot that is produced until the SPF supplier verifies that the chickens and the eggs produced by the chickens used to manufacture the vaccine lot were completely free of any disease. If the flock is found to have been infected, not only is the entire vaccine lot in question destroyed, the vaccine lots prior and subsequent to the vaccine lot are destroyed as well. This uncertainty adds a significant cost to the preparation of these vaccines.
Virus isolation diagnostic laboratories use embryonated SPF eggs to identify any of the aforementioned viruses. Since all of these viruses replicate well in embryonated eggs and the diagnostic laboratories do not need to prepare large quantities of virus, the expense of preparing CEF cells to detect or isolate the aforementioned viruses is unwarranted. However, VI diagnostic laboratories experience the same contamination problems experienced by vaccine manufacturers that rely on embryonated eggs. While several of the aforementioned viruses will replicate on mammalian cell lines, virus growth is poor and is limited to particular strains of virus adapted to grow on the cell line. Therefore, these cell lines have little utility in VI laboratories.
Vaccine manufacturers and VI diagnostic laboratories have long desired to replace their reliance on embryonated eggs with an immortal cell line. An immortal avian cell line would significantly reduced the cost of vaccine manufacture and the cost of VI diagnostic assays by eliminating the reliance on SPF embryonated eggs and the risk of contamination by adventitious agents either in the egg or during the preparation of primary cultures of CEF cells. An immortal avian cell line would enable for the first time the virus growing substrate to qualified by governmental regulatory agencies thereby avoiding many of the requirements currently in place. Both vaccine manufacturers and diagnostic laboratories would require the cell line to be avian in origin to ensure, in the case of VI diagnostic laboratories that the widest range of viruses could be detected, and in the case of vaccine manufacturers that the vaccine virus would be less likely to loss its potency. It is well known that viruses propagated in heterologous cells or cell lines can become incapable of replicating in the natural host. Furthermore, the viral antigens necessary for eliciting an effective immune response are altered during replication in the heterologous cell rendering vaccines made with the resulting virus ineffective. Vaccine manufacturers would also require the cell line to be completely virus free and non-oncogenic or non-tumorigenic in order to minimize the risk of inadvertently introducing viruses or transforming factors to the vaccine or vaccine virus and thereby into the vaccine recipient.
Many immortal cell lines derived from tissues of animal and human origin have been made. These cell lines have been established by chemical mutagenesis, viral transformation, directly from tumors, or have arisen unpredictably and spontaneously from primary cell cultures. However, cell lines that are virally transformed have no utility for vaccine production or VI diagnostic assays, cell lines established from tumors have little probability of being used for vaccines but may be used in some cases for VI diagnostic assays. Cell lines established by chemical mutagenesis or that occur "spontaneously" from primary cell cultures may be suitable for vaccines and VI diagnostic assays. However, some of these cell lines have subsequently been found to harbor retroviruses or have been found to be tumorigenic. The immortal cell line of the present invention contains neither any known virus nor is the cell line tumorigenic.
Current methods for propagating viruses that rely on embryonated eggs or primary cultures of CEF cells are recited next.